Selective R-cadherin antagonist and methods

ABSTRACT

An isolated peptide useful as a selective antagonist of mammalian R-cadherin comprises 3 to 30 amino acid residues, three contiguous residues of the peptide having the amino acid sequence Ile-Xaa-Ser; wherein Xaa is an amino acid residue selected from the group consisting of Asp, Asn, Glu, and Gln. Preferably Xaa is Asp or Asn. In one preferred embodiment the peptide is a cyclic peptide having 3 to 10 amino acid residues arranged in a ring. The selective R-cadherin antagonist peptides of the invention are useful for inhibiting the targeting of stem cells, such as endothelial precursor cells, to developing vasculature, for inhibiting R-cadherin mediated cellular adhesion, and for inhibiting retinal angiogenesis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application for Patent Ser. No. 60/467,188, filed on May 1, 2003, which is incorporated herein by reference.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with United States government support under Grants No. EY11254 and EY12598 from the National Institutes of Health. The United States government has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates generally to antagonists of mammalian cellular adhesion molecules. More specifically, the invention relates to selective peptide antagonists of mammalian R-cadherin (cadherin-4) and methods of inhibiting cellular adhesion and retinal angiogenesis therewith.

BACKGROUND OF THE INVENTION

The cadherin family of molecules consists of transmembrane glycoproteins that function in calcium dependent, selective cell-cell interactions. These molecules play important roles during embryonic development and tissue morphogenesis by mediating cell recognition and cell sorting. Subfamilies of cadherins (classic cadherins, protocadherins, desmocollins, and other cadherin-related proteins) are characterized by variable numbers of extracellular cadherin domains, a single transmembrane segment, and a single cytoplasmic domain. The so-called classic cadherins (i.e., E, P, N, and R-cadherin) reportedly have five tandemly repeated extracellular cadherin domains (EC1-EC5) that engage in preferentially homophillic interactions, and a highly conserved cytoplasmic tail that mediates adhesion specific intracellular signaling.

Cadherin mediated cell-cell adhesions occur as multiple cadherin molecules expressed on adjacent cells interact, leading to the formation of adherens junctions. According to the cadherin zipper model proposed by Shapiro et al. Nature 1995; 374(6520):327-37, cadherin molecules within the membrane of the same cell form tight parallel-strand dimers (i.e., so-called cis-dimers). As illustrated in FIG. 1, these cis-dimers then bind to cadherin dimers expressed on adjacent cells (i.e., trans-dimerization). Once a sufficient interaction is sustained, cadherin clustering can occur as more cadherin molecules are recruited to the site, leading to interdigitation of molecules from the two-cell surfaces. In this manner, relatively weak interactions can combine to form fairly strong cell-cell adhesions.

Upon initial cadherin adhesion, intracellular signals, transmitted through interactions of the cytoplasmic cadherin tails with α and β catenin molecules, lead to reorganization of the cytoskeleton. Although the association with actin filaments is not thought to affect homophillic binding, their association helps hold the cadherin molecules at the sites of interaction. In a symbiotic-type of relationship, cadherin clustering causes reorganization of the cytoskeleton and provides points of attachment at the membrane, which are important for cellular changes that occur upon the formation of adherens junctions. Meanwhile, association with the cytoskeleton holds cadherins at the sites of interaction and helps recruit new cadherin molecules, thus mediating cadherin clustering. Calcium plays an important role as a cofactor during cadherin clustering. Cadherin function is lost and molecules become more susceptible to protease degradation in solutions with insufficient concentrations of calcium ions (i.e., below about 2 mM). This is due to the requirement of calcium to stabilize the structure of cadherin molecules and provide proper orientation of adjacent cadherin interfaces.

Although it is reported that each of the five extracellular classical cadherin domains EC1 through EC5 plays an important role in mediating cadherin dimerization, mutational analysis has suggested that the majority of residues that form the dimerization interface are found within the N-terminal most cadherin domain (EC1) (Kitagawa, et al., Biochem. Biophys. Res. Commun., 2000; 271(2):358-63.). However, relatively little is known about the mechanisms of specific homodimerization between cadherin molecules.

Cadherins play a significant role during neuronal guidance and development of the central nervous system. Different subdivisions of the brain are reportedly defined by differential expression of cadherin types. Cadherins also play an important role in neural retinal development by specific expression within different regions of the developing retina. For example, during embryonic chick retina development, B-cadherin is reportedly only found in Müller glia, while certain populations of bipolar cells express R-cadherin (also known as cadherin-4). Amacrine cells and a subset of ganglion cells express cadherins 6B and 7. Within the inner plexiform layer of the retina, these same cadherins are only expressed in sublaminae associated with synapsin-I positive nerve terminals, suggesting that distinct expression profiles contribute to synapse formation between specific subpopulations of neurons during retina development. In the embryonic optic nerve, ganglion cell axon outgrowth is mediated by N-cadherin adhesion with R-cadherin-expressing glial cells.

Cadherin adhesion also plays a role in developmental retinal vascularization (Dorrell, et al. Invest. Ophthalmol. Vis. Sci. 2002; 43(11):3500-10). Disruption of R-cadherin adhesion during formation of the superficial vascular plexus results in the loss of complex vascular interconnections observed during normal vascular patterning. When R-cadherin adhesion is blocked during the subsequent formation of deep vascular layers, key guidance cues are lost causing the vessels to migrate past the normal deep vascular plexuses and into the photoreceptor layer.

The retina consists of well-defined layers of neuronal, glial, and vascular elements. Any disease or condition that alters the retinal layers even slightly, can lead to neuronal degeneration and significant visual loss. The retinal degeneration mouse (rd/rd mouse) has been investigated for over 70 years as a model for many diseases that lead to photoreceptor cell death. In the rd/rd mouse, photoreceptor degeneration begins during the first three weeks after birth as rod cells undergo apoptosis, attributed to a mutation in the β subunit of cGMP-dependent phosphodiesterase followed by cone photoreceptor death. Vascular atrophy within the retina is temporally associated with photoreceptor cell death in rd/rd mice as well. The vasculature appears to form in the normal characteristic fashion as three functional layers develop within the first three weeks. However, the vessels in the deep vascular layer begin to degenerate during the second week and by the end of the fourth postnatal week, dramatic vascular reduction is observed as the deep and intermediate plexuses nearly completely disappear.

A population of hematopoietic stem cells resides in the normal adult circulation and bone marrow, from which different sub-populations of cells can differentiate along lineage positive (Lin⁺HSC) or lineage negative (Lin⁻HSC) lineages. In addition, the present inventors have discovered that endothelial precursor cells (EPCs), capable of forming blood vessels in vitro and in vivo, are present within the Lin⁻HSC subpopulation. EPCs within the population of Lin⁻HSCs can target and stabilize the degenerating vasculature in rd/rd mice when injected intravitrally to the eyes of the mice. Intravitreally injected Lin⁻HSCs target astrocytes in the superficial vascular layer and are observed ahead of the endogenous developing vascular network when injected at postnatal day 2 (P2). As the endogenous vasculature reaches the periphery of the retina, where the Lin⁻HSCs have targeted, the cells are incorporated into new blood vessels, forming functional mosaic vessels with mixed populations of injected Lin⁻HSCs and endogenous endothelial cells. In addition, Lin⁻HSCs target the regions of deep and intermediate vascular layers of the retina before vascularization of these layers by endogenous endothelial cells had occurs. Incorporation of Lin⁻HSCs rescues the deep vasculature of rd/rd mice about 2 to about 3 fold over normal and control Lin⁺HSC injected mice. In addition, rescue of the deep vasculature prevents degradation of photoreceptors in the outer nuclear layer of the retina. However, as there is no evidence to suggest that these stem cells can undergo differentiation into retinal neurons or glial cells, the mechanism of neuronal protection remains unknown.

The targeting of Lin⁻HSCs to the astrocytes and deep vascular regions ahead of natural developmental vascularization suggests that the Lin⁻HSCs express cell-surface molecules that function in targeting, similar to targeting of the endogenous endothelial cells during development. R-cadherin adhesion plays an important role in endothelial cell targeting to astrocytes and vascular plexuses during developmental retinal angiogenesis.

R-cadherin has been identified and sequenced in a number of mammals. FIG. 2 depicts the amino acid sequence (SEQ ID NO: 1) of a human variant of R-cadherin preproprotein reported by Kitagawa et al. in the SWISS-PROT database as Accession No. NP 001785, version NP 001785.2, GI:14589893, the relevant disclosure of which is incorporated herein by reference. SEQ ID NO: 1 includes the amino acid sequence IDSMSGR (SEQ ID NO: 2) at positions 222-228.

FIG. 3 depicts the amino acid sequence (SEQ ID NO: 3) of another human variant of R-cadherin preproprotein reported by Tanihara et al. in the SWISS-PROT database as Accession No. P55283, version P55283, GI:1705542, the relevant disclosure of which is incorporated herein by reference. SEQ ID NO: 3 includes the amino acid sequence INSMSGR (SEQ ID NO: 4) at positions 222-228.

FIG. 4 depicts the amino acid sequence (SEQ ID NO: 5) of a murine (mus musculus) variant of R-cadherin preproprotein reported by Hutton et al. in the SWISS-PROT database as Accession No. NP 033997, version NP 033997.1, GI:6753376, the relevant disclosure of which is incorporated herein by reference. SEQ ID NO: 5 includes the amino acid sequence IDSMSGR (SEQ ID NO: 2) at positions 219-225.

Non-selective peptide antagonists of cadherins including the amino acid sequence His-Ala-Val (HAV) have been reported by Blaschuk et al. in U.S. Pat. No. 6,465,427, U.S. Pat. No. 6,3456,512, U.S. Pat. No. 6,169,071, and U.S. Pat. No. 6,031,072. Blaschuk et al. have reported both linear and cyclic peptide antagonists of cadherins, all of which are capable of antagonizing a number of types of cadherin molecules indiscriminately.

Selective peptide antagonists of N-cadherin, which comprise the amino acid sequence Ile-Asn-Pro (INP) have been reported by Williams et al., Mol. Cell Neurosci., 2000;15(5):456-64. While HAV peptides are non-specific cadherin antagonists, the INP peptide antagonists reported by Williams et al. are specific for N-cadherin and do not exhibit significant binding to other cadherin molecules such as R-cadherin.

Because of the differential distribution of cell adhesion molecules in various tissues in the body, there is an ongoing need for antagonists that are highly selective for specific cell adhesion molecules, in particular for antagonists that are selective for R-cadherin. The selective R-cadherin antagonist peptides of the present invention fulfill this need.

SUMMARY OF THE INVENTION

An isolated peptide useful as a selective antagonist of mammalian R-cadherin comprises 3 to 30 amino acid residues, three contiguous residues of the peptide having the amino acid sequence Ile-Xaa-Ser (IXS); wherein Xaa is an amino acid residue selected from the group consisting of Asp, Asn, Glu, and Gln. Preferably Xaa is Asp or Asn. In one preferred embodiment the peptide comprises at least seven amino acid residues and seven contiguous amino acid residues of the peptide have the amino acid sequence Ile-Xaa-Ser-Met-Ser-Gly-Arg (SEQ ID NO: 6), with Xaa being the same as defined above. The present invention also provides pharmaceutical compositions comprising the R-cadherin antagonist peptides in a pharmaceutically acceptable carrier.

In another preferred embodiment the peptide is a cyclic peptide having 3 to 10 amino acid residues arranged in a ring, three contiguous residues of the peptide having the amino acid sequence Ile-Xaa-Ser; wherein Xaa is an amino acid residue selected from the group consisting of Asp, Asn, Glu, and Gln. Preferably Xaa is Asp or Asn.

A preferred cyclic peptide has the formula:

wherein X¹ and X² are independently an amino acid residue or a plurality of up to 10 amino acid residues linked by pentide bonds; Y¹ and Y² are independently amino acid residues linked to one another by a disulfide bond and Xaa is an amino acid residue selected from the group consisting of Asp, Asn, Glu, and Gln.

A particularly preferred cyclic peptide has the amino acid sequence cyclic Cys-Ile-Xaa-Ser-Cys (SEQ ID NO: 7); wherein Xaa is an amino acid residue selected from the group consisting of Asp, Asn, Glu, and Gln, and the peptide ring is formed by a disulfide linkage between the two cysteine residues.

A method of inhibiting R-cadherin mediated cellular adhesion involves contacting R-cadherin expressing cells with an adhesion inhibiting amount of a selective R-cadherin antagonist peptide of the present invention. For example, retinal angiogenesis is inhibited by administering to a patient suffering from abnormal retinal vascular angiogenesis an angiogenesis inhibiting amount of a R-cadherin antagonist peptide of the present invention. Similarly, targeting of lineage negative hematopoietic stem cells to developing vasculature is inhibited by contacting the stem cells with a vasculature targeting inhibiting amount of a R-cadherin antagonist peptide of the present invention. Inhibiting targeting of Lin⁻HSCs, such as endothelial precursor cells, to developing vasculature is useful for treating diseases associated with abnormal vascular development such as age related macular degeneration and diabetic retinopathy.

BRIEF DESCRIPTION OF THE DRAWINGS

In the Drawings, FIG. 1 is a schematic representation of cadherin clustering and cadherin modulated cellular adhesion;

FIG. 2 depicts the amino acid sequence of a human variant of R-cadherin preproprotein (SEQ ID NO: 1), which includes the sequence IDSMSGR (SEQ ID NO: 2) at residues 222-228;

FIG. 3 depicts the amino acid sequence of a human variant of R-cadherin preproprotein (SEQ ID NO: 3), which includes the sequence INSMSGR (SEQ ID NO: 4) at residues 222-228;

FIG. 4 depicts the amino acid sequence of a murine variant of R-cadherin preproprotein (SEQ ID NO: 5), which includes the sequence IDSMSGR (SEQ ID NO: 2) at residues 229-225;

FIG. 5(A) illustrates the sequence homology within residues 24-92 of murine N-cadherin and various R-cadherins; note homologies between mammalian R-cadherins from human, mouse and rat, all of which comprise a sequence IDS or INS at residues 53-55, in contrast to chicken R-cadherin and mouse N-cadherin, which have the sequence IDP and INP, respectively at residues 53-55; (B) cyclic and linear peptides corresponding to residues within this region of murine and human R-cadherin and murine N-cadherin were synthesized along with the corresponding control peptides: cyclic CIDSC (SEQ ID NO: 8), cyclic CINPC (SEQ ID NO: 9), IDSMSGR (SEQ ID NO: 2), IDSASGR (SEQ ID NO: 10), INPASGQ (SEQ ID NO: 11), cyclic CSDIC (SEQ ID NO: 12), and cyclic CRADC (SEQ ID NO: 13); the partial cadherin sequences listed in FIG. 5(A) are, from top to bottom, murine N-cadherin (SEQ ID NO: 14), murine R-cadherin (SEQ ID NO: 15), rat R-cadherin (SEQ ID NO: 16), a human R-cadherin (SEQ ID NO: 17), another human R-cadherin (SEQ ID NO: 18), and chicken (gallus gallus) R-cadherin (SEQ ID NO: 19);

FIG. 6(A) depicts photomicrographs demonstrating aggregation of L-cells expressing R-cadherin and N-cadherin; (B) is a bar graph of percent aggregation of L-cells mediated by R and N-cadherins in the presence and absence of calcium;

FIG. 7 demonstrates stable transfection of L-cells with R-cadherin and N-cadherin; (A) R-cadherin immunoblot; (B) N-cadherin immunoblot; (C-E) photomicrographs of stained L-cells demonstrating expression of R-cadherin (C), and N-cadherin (D), compared to cells expressing neither R nor N-cadherin (E);

FIG. 8 graphically illustrates selective inhibition of aggregation of cadherin expressing L-cells by IDS containing peptides, which bind to R-cadherin expressing cells, compared with INP containing peptides, which bind to N-cadherin expressing cells;

FIG. 9 illustrates selective inhibition of mouse retinal vascularization after intravitreal injection of cyclic CIDSC (SEQ ID NO: 8), compared with cyclic CINPC (SEQ ID NO: 9); (A) depicts photomicrographs of rd/rd mouse retinas at the P2 stage of development; (B) depicts photomicrographs of rd/rd mouse retinas at the P7 stage of development; (C) is a bar graph of superficial vascularization; (D) is a bar graph of deep vascularization;

FIG. 10 illustrates the results of flow cytometry analysis of R-cadherin expression in hematopoietic stem cells (HSC);

FIG. 11 depicts cross-sectional photomicrographs of rd/rd mouse retinas treated with various peptides of the invention and control peptides; and

FIG. 12 illustrates the blocking of Lin⁻HSC targeting of developing retinal vasculature by the R-cadherin antagonist peptides of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As used herein and in the appended claims, the term “cyclic peptide” refers to molecules comprising a plurality of amino acids linked together in a chain by peptide linkages, the ends of the chain being joined together to form a ring of amino acid residues. The cyclic peptide can be joined together by a peptide bond, a disulfide linkage between two amino acid residues such as cysteine residues, or by any other suitable linking group. Nonpeptidal linking groups can be any chemical moiety that can react with functional groups at each end of the peptide chain to form a link therebetween. For example, two ends of a peptide chain can be linked together by a non-protein amino acid such as 3-aminobutyric acid or by a disulfide formed from nonpeptidal thiol groups such as a thioglycolic amide at the amino terminal end and amide formed from 2-aminoethane thiol at the carboxy terminal end, for example.

As used herein and in the appended claims, the term “pharmaceutically acceptable” and grammatical variations thereof, in reference to carriers and other excipients, means that the materials are capable of administration to a human patient without the production of undesirable physiological side effects such as retinal or ocular irritation, nausea, dizziness, blurred or impaired vision, cytotoxicity, and the like.

The term “amino acid” as used herein and in the appended claims refers generally to any alpha amino acid. Preferably, the peptides of the present invention comprise the 21 amino acids encoded by the genetic code, although modified amino acid residues can also be included. The amino acids can be in the L, D, or D,L form. Preferably, the peptides of the present invention comprise L-form amino acids. To minimize the likelihood of proteinase degradation in vivo, the administered peptides of the present invention can include one or more D-form amino acid residues.

An isolated peptide, which is a selective antagonist of mammalian R-cadherin comprises 3 to 30 amino acid residues, three contiguous residues of the peptide having the amino acid sequence Ile-Xaa-Ser. Xaa is an amino acid residue selected from the group consisting of Asp, Asn, Glu, and Gln. Preferably Xaa is Asp or Asn. The R-cadherin antagonist peptide of the present invention can be linear or cyclic.

The selective R-cadherin antagonist peptides of the present invention mimic the Ile-Asp-Ser and Ile-Asn-Ser sequences found in the EC1 domain of mammalian R-cadherin, but not in other cadherin molecules. Peptides comprising the Ile-Xaa-Ser sequence can bind to and antagonize mammalian R-cadherin molecules. Xaa preferably is an aspartic acid residue (Asp) or an asparagine residue (Asn), to match the naturally occurring sequences in mammalian R-cadherin molecules. Glutamic acid (Glu) and glutamine (Gln) residues are also suitable for Xaa, due to their chemical similarity to Asp and Asn, respectively.

In one preferred embodiment, the peptide comprises at least seven amino acid residues and seven contiguous amino acid residues of the peptide have the amino acid sequence Ile-Xaa-Ser-Met-Ser-Gly-Arg (SEQ ID NO: 6). Xaa is an amino acid residue selected from the group consisting of Asp, Asn, Glu, and Gln. Preferably Xaa is Asp or Asn.

In another preferred embodiment the peptide is a cyclic peptide having 3 to 10 amino acid residues arranged in a ring, three contiguous residues of the peptide having the amino acid sequence Ile-Xaa-Ser; wherein Xaa is an amino acid residue selected from the group consisting of Asp, Asn, Glu, and Gln, as described above. Preferably Xaa is Asp or Asn.

A preferred cyclic peptide having five amino acids arranged in a ring has the formula:

wherein X¹ and X² are independently an amino acid residue or a plurality of up to 10 amino acid residues linked by peptide bonds; Y¹ and Y² are independently amino acid residues linked to one another by a disulfide bond and Xaa is an amino acid residue selected from the group consisting of Asp, Asn, Glu, and Gln. Preferably Y¹ and Y² are both cysteine residues linked together by a disulfide bond (i.e., a cystine residue).

A particularly preferred cyclic peptide has the amino acid sequence cyclo Cys-Ile-Xaa-Ser-Cys (SEQ ID NO: 7); wherein Xaa is an amino acid residue selected from the group consisting of Asp, Asn, Glu, and Gln, and the ring is formed by a disulfide linkage between the two cysteine residues. Preferably Xaa is Asp or Asn.

A method of inhibiting R-cadherin mediated cellular adhesion involves contacting R-cadherin expressing cells with an adhesion inhibiting amount of a R-cadherin antagonist peptide of the present invention. The cells can be contacted in vivo with the peptide antagonist by administering a cellular adhesion inhibiting amount of the antagonist to a mammal suffering from a disease or condition that is treatable by inhibiting R-cadherin mediated cellular adhesion (e.g., retinal diseases characterized by abnormal vascular proliferation). For example, a human patient suffering from age related macular degeneration or diabetic retinopathy can be beneficially treated with a selective R-cadherin antagonist peptide of the present invention. Preferably the antagonist is administered as a pharmaceutical composition comprising the antagonist and a pharmaceutically acceptable carrier therefor.

For the selective targeting or antagonism of R-cadherin the peptides and compositions of the present invention may be administered in a therapeutically effective amount parenterally, orally, by inhalation, or topically in unit dosage form together with pharmaceutically acceptable carriers, vehicles, and adjuvants. The term “parenteral,” as used herein, includes intravenous, subcutaneous, intramuscular, intrasternal, intraocular (e.g. intravitreal), and intraperitoneal administration, as well as administration by infusion techniques.

Any suitable route of administration can be utilized, and the pharmaceutical composition including a selective R-cadherin antagonist peptide of the present invention is administered in a dose effective for the intended treatment. Therapeutically effective amounts required to treat a particular medical condition, or inhibit the progress thereof, are readily determined by those skilled in the art using preclinical and clinical studies known in the medical arts.

The term “therapeutically effective amount,” as used herein, refers to that amount of active ingredient that elicits the biological or medical response of a tissue, system, animal or human, sought by a clinician or a researcher.

The term “inhibit,” as used herein, refers to a slowing, interruption, or stoppage of the medical condition or a biochemical interaction, but does not necessarily indicate a total elimination of the condition or complete interruption of the interaction. A prolonged survivability of a patient or prolonged reduction in the severity of symptoms, in and of itself, indicates that the medical condition is beneficially controlled (i.e., inhibited).

The dosage regimens for the present R-cadherin antagonist peptides and compositions containing the same, are based on several factors such as the age, weight, sex, and type of medical condition of the patient, the severity of the condition, the route of administration, and the antagonist activity of the particular peptide antagonist employed. The dosage regimen may vary depending upon the aforementioned factors. Dosage levels on the order of about 0.01 milligram to about 1000 milligrams per kilogram of body weight are useful for inhibiting retinal angiogenesis, for example. Preferred dosage levels are in the range of about 0.01 milligram to about 100 milligrams per kilogram of body weight.

For administration by injection, a peptide-containing composition embodying the present invention is formulated with a pharmaceutically acceptable carrier such as water, saline, or an aqueous dextrose solution. For injection, a typical daily dose is about 0.01 milligram to about 100 milligrams per kilogram of body weight, iniected daily as a single dose or as multiple doses depending upon the aforementioned factors.

Pharmaceutical compositions of the present invention comprising a selective R-cadherin antagonist peptide of the invention and a pharmaceutically acceptable carrier can also include pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients that can be included in the pharmaceutical compositions of the present invention include, for example, physiologically tolerable surfactants, solvents, buffering agents, preservatives, and the like, which are well known in the art.

To inhibit retinal angiogenesis, for example, a patient suffering from abnormal retinal vascular proliferation is administered a therapeutically effective amount of a R-cadherin antagonist peptide of the present invention. The administered peptide selectively binds to R-cadherin in the retina, thus disrupting and inhibiting angiogenesis therein. Preferably the peptide antagonist is administered by intravitreal injection.

Targeting of Lin⁻HSCs to developing vasculature is inhibited by contacting the stem cells with a vasculature targeting inhibiting amount of a selective R-cadherin antagonist peptide of the present invention. Inhibiting targeting of Lin⁻HSCs, such as endothelial precursor cells, to developing vasculature is useful for treating diseases associated with abnormal vascular development such as age related macular degeneration and diabetic retinopathy. Preferably the Lin⁻HSCs are contacted in vivo by administering the present R-cadherin antagonist peptides to a mammal, such as a human, suffering from a vascular proliferative disease or condition.

The following non-limiting examples are provided to further illustrate the various aspects of the invention. One of skill in the art will recognize that modifications of the examples and illustrated embodiments disclosed herein can be made without departure from the spirit and scope of the invention.

EXAMPLE 1 Peptide Synthesis

The peptides of the present invention and various control peptides were synthesized by The Scripps Research Institute Protein and Nucleic Acids core facility using the solid phase synthesis method, and were purified to the highest grade possible (>95% pure) as analyzed by HPLC analysis. The sequences of the peptides were analyzed by mass spectrometry to ensure synthesis of the correct peptides. All peptides were amide blocked at the amino terminus and acetylated at the carboxy terminus. The cyclic peptides were prepared with cysteine residues at the amino and carboxy terminal ends to create a disulfide tether and form a ring containing five amino acid residues. FIG. 5 (B) illustrates the peptides prepared: cylic CIDSC (SEQ ID NO: 9), cylic CINPC (SEQ ID NO: 9), IDSMSGR (SEQ ID NO: 2), IDSASGR (SEQ ID NO: 10), INPASGO (SEQ ID NO: 11), cylic CSDIC (SEQ ID NO: 12), and cylic CRADC (SEQ ID NO: 13).

EXAMPLE 2 L-Cell Transfections

Mouse R-cadherin and N-cadherin plasmids were generous gifts from Dr. Masatoshi Takeichi (Kyoto University, Japan). The plasmids were sub-cloned into pDsRed2 N1 vectors (Clontech) to encode for fusion proteins with Red Fluorescent Protein (RFP) attached to the C-terminal end of the cadherin molecules. L-cells (mouse fibroblast L929 cells, ATCC #CRL-2148) were stably transfected with either R or N-cadherin pDsRed2 N1 using the Calcium Phosphate Transfection System (Life Technologies) according to the manufacturer's protocol. After screening by growth in media supplemented with Geneticin (700 μg/mL G418 Geneticin, Gibco BRL), positive clones were selected. Cells were examined for expression of RFP, and were tested for cadherin expression by immunoblotting and immunofluorescence staining. FIG. 6 illustrates the aggregation of L-cells expressing R and N-cadherins. FIG. 6(A) shows photomicrographs of R-cadherin (left) and N-cadherin (middle) expressing L-cells aggregating in calcium containing media, compared to non-transfected L-cells, which did not aggregate. FIG. 6(B) is a bar graph illustrating the percentage of aggregation of the cells shown in FIG. 6(A). N-cadherin and R-cadherin transfected cells trypsinized in a buffer containing about 5 mM calcium chloride (labeled TC) formed large cell clusters, whereas endogenous L-cells showed little aggregation in the calcium containing buffer. Cells that were trypsinized with EDTA in a calcium free buffer (labeled TE) showed little aggregation, regardless of whether the cells were transfected with cadherins or not.

EXAMPLE 3 Cell Culture and Immunofuorescence

Transfected or wild type L-cells were grown in Modified Eagles Medium (MEM) supplemented with Earl's Basic Salt Solution, 2 mM Glutamax, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, and 10% fetal bovine serum. Transfected cell lines were grown in media supplemented with about 700 μg/mL G418 (all media reagents were from Gibco BRL). For immunofluorescence, the cells were grown to about 75% confluency on glass cover slips. The cells were fixed in 4% paraformaldehyde for about one-half hour followed by blocking with 5% normal goat serum and 5% fetal bovine serum in 1× phosphate buffered saline (PBS). Goat polyclonal antibodies against R-cadherin or N-cadherin (Santa Cruz) were used at 1:200 dilution, and fluorescence was conferred by incubation with Alexa488 labeled anti-goat IgG secondaries (Molecular Probes). Images were created using a Radiance 200 fluorescence confocal microscope (BioRad). For immunoblot analysis, cells were lysed in buffer containing 1% Triton X-100. About 50 μg of total cell lysate was added to each lane of an 8% polyacrylamide gel and proteins separated by electrophoresis. Monoclonal antibodies (1:1000, BD Biosciences) specific for N-cadherin or R-cadherin were used to visualize the corresponding bands.

FIG. 7(A) shows an immunoblot of native L-cells and L-cells transfected with R-cadherin and N-cadherin and stained with R-cadherin antibody. Only the R-cadherin transfected cells exhibited significant levels of R-cadherin expression. FIG. 7(B) shows an immunoblot of native L-cells and L-cells transfected with R-cadherin and N-cadherin and stained with N-cadherin antibody. Only the N-cadherin transfected cells exhibited significant levels of N-cadherin expression. FIG. 7(C) is a fluorescence photomicrograph of R-cadherin expressing cells labeled with fluorescent cadherin antibodies, demonstrating cell surface expression of only the R-cadherin molecules. FIG. 7(D) is a fluorescence photomicrograph of N-cadherin expressing cells labeled with fluorescent cadherin antibodies, demonstrating cell surface expression of only the N-cadherin molecules. FIG. 7(E) is a fluorescence photomicrograph of native L-cells exposed to fluorescent cadherin antibodies, but showing no cell surface expression of cadherin molecules of either type.

EXAMPLE 4 Aggregation Assay

L-Cells were grown to near confluency followed by trypsinization with 0.01% trypsin +5mM CaCl₂ and no EDTA (TC) or 0.01% trypsin with 0.1 mM EDTA and no calcium (TE). The cells were collected and washed, followed by resuspension in Hanks buffer solution (HBSS) +1% BSA with (TC) or without (TE) 5mM CaCl₂. Cells were incubated at 37° C. in 0.5 mL solution at 2×10⁵ cells per well of a 24 well plate with rocking at about 60-70 rpm with varying peptide concentrations. All assays were performed in triplicate. The extent of cellular aggregation was represented by the ratio of the total particle number after 2 hours of incubation (N_(2hr)) to the initial particle number (N₀). Particles were counted on a hemocytometer using the sum of 8 separate 20 μL counts/well, before (N₀) and after (N_(t)) incubation. The results are illustrated in FIG. 8.

EXAMPLE 5 Treatment of Mice by Intravitreal Injection of Peptides

Peptides were dissolved in PBS +10% DMSO to a concentration of 10 mM. About 0.5 μL or 1.0 μL of 10 mM peptide solution was injected into the vitreal cavity of 2 day or 7 day-old mice respectively. At P5 or P11, the retinas were dissected as described and the vessels and astrocytes visualized by immunohistochemistry. Quantification of peripheral vascularization, vascular length, and vascular area during superficial vascular formation was achieved by imaging injected retinas under the same microscopy settings. Numbers were then generated using LASERPIX® software (BioRad) with non-injected control littermates used for baseline normalization of the extent of retinal vascularization. Quantification of the effect on deep vascular formation was achieved by focusing anterior to the normal deep vascular plexus using confocal microscopy, and counting the numbers of vessels that had migrated into the photoreceptor layer. The results are presented in FIG. 9.

EXAMPLE 6 Stem Cell Isolation and Enrichment

Bone-marrow cells were isolated from adult transgenic mice in which enhanced GFP was fused to the p-actin promoter (ACTbEGFP, the Jackson Laboratory, Bar Harbor, Me.). Monocytes were then collected by density gradient separation using Histopaque (Sigma) and labeled with biotin-conjugated lineage panel antibodies (CD45, CD3, Ly-6G, CD11, TER-119, Pharmingen, San Diego, Calif.) for Lin⁻ selection. Lin⁺ and Lin⁻ cells were separated using a magnetic separation column (MACS, Miltenyi Biotech, Auborn, Calif.). Since it was determined that CD31⁻ cells represent a better control of a non-functional subpopulation of HSCs as determined by vascular targeting, CD31⁻ cells were isolated from the monocytes by MACS sorting using CD31 antibodies and used as a negative control for the functional Lin⁻HSCs. HSCs from wild type mice were analyzed for the expression of R-cadherin by labeling cells with anti-R-cadherin antibodies (sc-6456, Santa Cruz Biotech) and Alexa-488 labeled donkey anti-goat secondaries (Molecular Probes), and using a FACS calibur (Beckton Dickinson, Franklin Lakes, N.J.) flow cytometer. The results are presented in FIG. 10.

EXAMPLE 7 HSC Cell Incubations, Injections, and Quantification

Lin⁻HSCs were incubated with 100 nM of R-cadherin blocking antibody (SC-6456, Santa Cruz Biotech), or pre-immune goat IgG in phosphate buffered saline solution for about 1 hour at about 37° C. prior to injection. Intravitreal injections into P6 eyes were performed using 0.5 μL of HSC solution. Retinas were then examined at P12 by whole mount or sections. Targeting of the Lin⁻HSCs was quantified by counting the total number of stem cells within the retina using eight different fields of view per retina: left, right, top, and bottom quadrants (¾ distance to the retina periphery), two intermediate quadrants (¼-½ distance to the periphery), the injection site, and the optic nerve head region. These cells were characterized by their localization to the superficial, intermediate, or deep layers, or by the lack of targeting (cells that lie at the back of the photoreceptor layer). The number of non-targeted cells within the photoreceptor layer is given as a percentage of the total number of stem cells observed. The results are presented in FIG. 11 and FIG. 12.

Discussion

The selective R-cadherin antagonist peptides of the present invention act as peptide mimetics of key recognition motifs of R-cadherin (i.e, the IDS and INS sequences found in mammalian R-cadherins). Without being bound by theory, the present antagonist peptides likely block the adhesion function of R-cadherin molecules by competitive interaction with the EC1 domains of R-cadherin molecules on cell surfaces. The present antagonist peptides are useful for the study of molecular functions, and for the treatment of cellular adhesion-related diseases, and are generally are more diffusible within a tissue upon in vivo injection than antibody-based antagonists.

Tissue morphogenesis during the development of most tissues, including retinal neural tissue, involves the selective binding of cell-cell adhesion molecules. This binding selectivity allows similarly differentiated cells to organize together, and prevents cell types from invading incorrect tissue structures. However, despite the extensive studies on cadherin properties and function, particularly N- and E-cadherins, a general mechanism accounting for cadherin specificity has not yet emerged. The present R-cadherin antagonist peptides selectively interact with mammalian R-cadherin molecules without significant binding to other cadherin classes. These peptides contain the IDS sequence (or its homologs INS, IES, and IQS), which corresponds to a region within cadherin domain EC1, residues 53-55 of SEQ ID NO: 17 and 18, where important interactions within the adhesion interface are reportedly located based on structural, mutational, and sequence homology analysis.

Without being bound by theory, the IDS motif is thought to make direct contacts with the VDI sequence from an adjacent cadherin molecule at the adhesion interface. Because residues 53-55 of cadherins appear to be required for transdimerization, and because unlike other adhesion-important regions this short sequence of amino acids was not conserved amongst classical cadherin family members, this region acts as a determinant for cadherin specificity. Indeed, cyclic IDS (CIDSC, SEQ ID NO: 8) selectively inhibits R-cadherin mediated cellular aggregation, while the corresponding N-cadherin counterpart, cyclic INP (CINPC, SEQ ID NO: 9) selectively inhibits N-cadherin mediated aggregation. N-cadherin and R-cadherin are the most homologous of the cadherin family members. In fact, although all cadherins, including R and N-cadherin prefer to interact in a homophillic manner, R- and N-cadherin are the only two classical cadherin family members where functional heterodimers have been observed. Thus, it is highly unlikely that cyclic IDS and cyclic INP would have distinct functional properties for N and R-cadherin, but overlapping properties for any other cadherin member. These studies demonstrate that the IXS motif (where X is D, N, E, or Q) (i.e. corresponding to R-cadherin residues 53-55, and homologs thereof), plays an important role in mediating homoassociation of R-cadherin molecules. It is likely, based on the specificity of IXS for R-cadherin and INP for N-cadherin, that correspondingly placed residues in other cadherin family members (e.g., the IER motif of E-cadherin and the IEK motif of P-cadherin) also impart specificity to the other classical cadherins as well.

Previous studies have shown that antibodies against R-cadherin disrupt retinal vascularization in vivo. Vascularization of the superficial plexus likely was disrupted due to the interruption of R-cadherin mediated guidance cues relayed by astrocytes lying ahead of the endothelial cells. R-cadherin expression is also observed in the regions where the deep vascular plexuses are subsequently located, just ahead of vascular invasion. R-cadherin molecules within these regions are thought to guide endothelial cells to the correct vascular plexus, since injection of R-cadherin blocking antibodies causes vessels to bypass the normal vascularized layers.

Similar vascular phenotypes have now been generated by injection of cyclic IDS (CIDSC, SEQ ID NO: 8) during both superficial and deep retinal vascularization. Since cyclic IDS selectively disrupted R-cadherin mediated aggregation to a significant extent in vitro (i.e., without significant disruption of N-cadherin mediated aggregation), it is likely that the in vivo vascular phenotype was generated by high affinity interactions of cyclic IDS with R-cadherin, as well. In addition, injection of cyclic INP (CINPC, SEQ ID NO: 9), which was an effective inhibitor of N-cadherin mediated aggregation but not R-cadherin aggregation in vitro, did not result in a significant retinal vascular phenotype. Together, these results confirm a specific role for R-cadherin during vascular guidance.

The design of the selective R-cadherin antagonist peptides of the present invention was based on structural, biochemical, and mutational analysis of various members of the classical cadherin family. Tryptophan-2 is known to be important for cadherin function along with the HAV sequence at amino acid residues 79-81 of N-cadherin and R-cadherin. In fact, linear and cyclic peptides containing the HAV sequence reportedly block N-cadherin mediated neurite outgrowth in vitro. However, these sequences are absolutely conserved across all cadherin molecules and therefore carinot confer specificity of binding. Other residues must also make important contacts within the dimerization interface, with some non-conserved residues important for cadherin recognition. Attention was focused on residues within the amino terminal cadherin repeat (EC1). The majority of contact-important residues were localized to three regions, amino acids 35-45, amino acids 53-59, and amino acids 79-86. Residues 53-59, contained the majority of these cadherin specific residues potentially important in the formation of the dimerization interface. Of these, residues 53-55 were of particular significance. Thus, peptide mimetics were designed from this region to optimize the probability that R-cadherin specific peptides would be produced. Similar peptides against sequences from mouse N-cadherin, the most closely related cadherin family member, and other control peptides were designed and used for comparative analysis.

Cadherin Mediated Aggregation

Mouse fibroblast cells (lineage L929), commonly referred to as L-cells, were chosen because they are known to contain no endogenous cadherin expression. R-cadherin stable transfectants were created and used to test the effects of the designed peptides on R-cadherin mediated aggregation. N-cadherin stable transfectants were also created and used to evaluate the peptides for cadherin selectivity, based on their effects on N-cadherin mediated aggregation. Immunoblot analysis detected high levels of R-cadherin and no N-cadherin expression in R-cadherin clone 8 (R-cad8), while high levels of N-cadherin expression and no R-cadherin expression was found in N-cadherin clone 3 (N-cad3), as shown in FIG. 7. Immunofluorescence confirmed the expression of the appropriate cadherin in these chosen clones. When tested in the aggregation assay, the morphology of the transfected cell lines was altered as a result of cadherin transfection. While the parent (i.e., non-transfected) L-cells remained dissociated as single cell particles, the mouse R-cadherin and N-cadherin transfectants formed large, calcium-dependent (TC buffer), cell clusters due to tight intercellular associations, as shown in FIG. 6(A). Cadherin mediated aggregation clusters were eliminated by initial trypsinization of the cells with EDTA solution and aggregation in calcium-free buffer (TE buffer), as shown in FIG. 6(B).

Peptide Effects on Cell Aggregation

Peptides were added at varying concentrations to the aggregation wells to test their effectiveness at blocking cadherin mediated adhesion. Cyclic IDS inhibited R-cadherin mediated aggregation with an IC₅₀ of around 300 μM. The linear peptide IDSMSGR (SEQ ID NO: 2), also blocked R-cadherin mediated adhesion. However, its effectiveness (IC₅₀˜900 μM) was about 3 times lower than that of cyclic IDS. As the cyclic peptides also proved to be much more soluble and easier to work with than the linear peptides, further emphasis was focused solely on analysis of cyclic peptides (FIG. 8(A)). The effects of cyclic IDS were specific for R-cadherin, as little effect on N-cadherin aggregation was observed. In contrast, the corresponding N-cadherin specific sequence, cyclic INP, inhibited N-cadherin mediated aggregation with an IC₅₀ just below 300 μM (FIG. 8(B)), similar to the effects of cyclic IDS on R-cadherin aggregation. Cyclic INP had little effect on R-cadherin mediated aggregation.

The other control peptides, cyclic RAD (CRADC, SEQ ID NO: 13) and cyclic SDI (CSDIC, SEQ ID NO: 12) had little effect on either R-cadherin or N-cadherin mediated aggregation. A cyclic HAV peptide (CHAVC, SEQ ID NO: 20), already known to be effective at blocking adhesion mediated by any classical cadherin molecules, was tested as a comparison. In our assay, cyclic HAV blocked R-cadherin and N-cadherin mediated aggregation with IC_(50S) between 150 and 200 μM. Thus, cyclic IDS and cyclic INP selectively blocked R or N cadherin adhesion respectively, with only slightly lower affinities than the non-specific pan cadherin blocking peptide. Previous studies using antibodies against R-cadherin were shown to disrupt normal retina developmental vascularization. These antibodies were also effective at disrupting cadherin mediated aggregation in our assay system with an IC₅₀ of around 10 nM, as shown in FIG. 8(C).

Effects of Peptides on Retinal Vascularization

Peptides were injected into the vitreal cavity of postnatal mouse eyes. When cyclic IDS or cyclic HAV peptides were injected into two-day old mouse eyes, and the resulting vasculature was examined three days later at postnatal day 5 (P5), vascular formation was disrupted with results similar to those observed by antibody injections. These retinas were characterized as having less extensive peripheral vascularization, and fewer interconnecting vessels within the vascularized regions compared to normal, non-injected littermate controls. Overall, vascularization of the superficial layer was cut in half by R-cadherin blocking peptides while retinas with N-cadherin specific cyclic INP injections were relatively normal (see FIGS. 9(A-C)).

Selective R-cadherin antagonist peptides of the present invention disrupted normal vascularization of the deep retinal layers as well. When cyclic IDS peptide was injected at P7, just before vessels of the superficial vascular network dive and begin formation of the deep vascular plexus, the resultant P11 vasculature was characterized by numerous vascular sprouts that had migrated past the normal deep vascular plexus and into the avascular photoreceptor layer. Again, this is similar to the effects observed previously when R-cadherin antibodies were injected. In contrast, the deep vascular plexus of eyes injected with cyclic INP peptide formed normally, as shown in FIGS. 9(B and D).

R-Cadherin is Expressed by Lin⁻HSCs

Hematopoietic stem cell (HSC) expression of R-cadherin was analyzed to determine if R-cadherin cell adhesion molecules were expressed at the cell surface of functionally targeting cells. Using flow cytometry analysis, R-cadherin was expressed at the cell surface of nearly 80% of the Lin⁻ subpopulation of HSCs while only 30% of the Lin⁺ cells express R-cadherin (FIG. 10). Based on the relative fluorescence intensities between the two cell populations, it is likely that the Lin⁻ cells also express higher concentrations of R-cadherin at their cell surface, than the small portion of R-cadherin positive Lin⁺ cells. Thus, the majority of cells within the subpopulation that functionally targets the retinal vasculature express R-cadherin while most of the cells from the non-targeting subpopulation do not. Interestingly, a different subpopulation of HSCs that are CD31, CD34, and Mac1 negative and have no targeting function at all, contained even fewer R-cadherin expressing cells.

R-Cadherin Blocking Antibodies and Peptides Disrupt HSC Targeting

To examine the degree to which R-cadherin cell-cell adhesion functions in targeting of HSCs to the distinct retinal vascular layers, Lin⁻HSCs were blocked with R-cadherin specific, blocking antibodies prior to injection. Six days after injection, normal Lin⁻HSCs are only found localized to the three vascular layers: 1) the superficial vascular plexus localized within the ganglion cell layer, 2) the deep vascular plexus localized near the outer plexiform layer, and 3) the intermediate layer localized at the front edge of the inner nuclear layer. FIG. 11(A) shows cross-sections of retinas after injection of normal Lin⁻HSCs (left), Lin⁻HSCs incubated with adhesion blocking R-cadherin antibodies (middle) and Lin⁻HSCs incubated with pre-immune goat IgG (right).

When the Lin⁻HSCs were pre-incubated with anti-R-cadherin antibodies prior to injection, many of these cells lost their ability to target correctly, while cells pre-incubated with pre-immune IgG function similar to non-blocked HSCs. Targeting to the deep and intermediate vascular layers appears to be especially affected by blocking R-cadherin adhesion as relatively few R-cadherin blocked HSCs were found localized within these regions. The cells localized to the superficial vascular plexus also appeared less organized and were not co-localized with the endogenous vasculature to the same extent as normal Lin⁻HSCs or those pre-incubated with pre-immune IgG.

Many of the Lin⁻HSCs pre-incubated with R-cadherin antibody, migrated through the retina past all three vascular layers, and attached themselves to the outer edge of the photoreceptors near the RPE layer. Almost half of the R-cadherin blocked HSCs were found at the outer edge of the photoreceptor layer (FIG. 11(B)). In comparison, control retinas injected with HSCs pre-incubated with pre-immune IgG only had 15% of the HSCs mistargeted to this region. A large portion of the mistargeted cells from the control reginas were found near the injection site, and can likely be attributed to cells that were released subretinally as the needle was being removed. When the injection site was excluded from the calculation, the number of mistargeted pre-immune IgG incubated HSCs was reduced to 10%. Since almost no Lin⁻HSCs are observed within this “extra deep” layer normally, this small percentage of mistargeted control IgG incubated HSCs can likely be attributed to the fact that the pre-immune IgG was able to bind to about 10% of the Lin⁻cells (FIG. 10). These bound IgG molecules may non-specifically prevent normal adhesion simply due to steric hindrances. However, the difference between the number of mistargeted cells due to specific R-cadherin blocking, and non-specific IgG blocking, is significant.

FIG. 12(A) shows confocal images through z-planes of the three vascular plexuses and the outer edge of the photoreceptor layer. Normal targeting within correct vascular plexuses and along endogenous vessels was observed by Lin⁻HSCs blocked with pre-immune goat IgG. Blocking R-cadherin adhesion caused many Lin⁻HSCs to be localized at the outer edge of the photreceptor layer, and cells targeted to the normal vascular plexuses tended to clump together and were not localized along endogenous vessels. FIG. 12(B) is a bar graph demonstrating that the percentage of mistargeted cells relative to the entire population of HSCs within the retina was significantly greater for the R-cadherin blocked population of Lin⁻HSCs (P values <0.01).

The foregoing description is to be taken as illustrative, but not limiting. Still other variants within the spirit and scope of the present invention will readily present themselves to those skilled in the art. 

1. An isolated peptide composition consisting of SEQ ID NO:
 8. 2. A pharmaceutical for inhibiting retinal angiogenesis comprising the peptide of claim 1 and a pharmaceutically accptable excipient in a pharmaceutically acceptable carrier therefor. 